骨骼肌胰岛素受体底物-1及其丝氨酸磷酸化与酪氨酸磷酸化在感染大鼠胰岛素抵抗中的作用

【摘要】目的研究不同程度感染大鼠骨骼肌胰岛素受体底物-1（IRS-1）及其丝氨酸（Ser^307）磷酸化和酪氨酸（Tyr）磷酸化在胰岛素抵抗（IR）中的作用。方法SD大鼠随机分为对照组、10％组和30％组。10％组和30％组大鼠用盲肠结扎穿孔制造感染模型,结扎长度分别为总长度的10％和30％,对照组为假手术组。3组大鼠分别在术前和术后各时间点取空腹血和后腿腓肠肌后处死。测定血浆空腹血糖（FPG）、空腹血胰岛素（FINS）、TNF-α和IL-6浓度,计算IR指数（IgIRI）,同时检测IRS-1及其Tyr磷酸化和Ser^307磷酸化水平。结果3组大鼠生存曲线之间,均差异有统计学意义（P〈0．01）。术后10％组和30％组TNF-α和IL-6均明显高于对照组（均P〈0．01）,其中30％组更高（均P〈0．01）。术后10％组和30％组FPG、FINS和IgIRI在各时间点均明显高于对照组（均P〈0．01）,而30％组则明显高于10％组（P〈0．01）,术后FINS升高程度要明显高于FPG。对照组和30％组IRS-1均呈阳性位于骨骼肌细胞质内;对照组IRS-1Tyr磷酸化呈阳性,而30％组仅呈散在阳性;对照组IILS-1Ser^307磷酸化呈阴性,而30％组呈强阳性。半定量分析显示30％组IRS—1在术后各个时间点与对照组相似（均P〉0．05）,IRS-1Tyr磷酸化在30％组术后各时间点明显低于对照组（均P〈0．01）,IRS-1Ser^307磷酸化在术后各时间点明显高于对照组（均P〈0．01）。IgIRI与IRs-1 Tyr磷酸化呈显著负相关（r=-0．957,P〈0．01）,与IRS-1Ser^307磷酸化呈显著正相关（r=0．955,P〈0．01）。结论感染状态下骨骼肌细胞内IRS-1的含量不变,而是其Tyr磷酸化降低,同时Ser^307磷酸化增强,其变化程度与机体胰岛素抵抗程度密切相关。

Effects of insulin receptor substrate-1 and its serine phosphorylation and tyrosine phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis: experiment with rats

OBJECTIVE: To investigate the effects of insulin receptor substrate (IRS)-1 and its serine (Ser)(307) phosphorylation and tyrosine (Tyr) phosphorylation on insulin resistance in skeletal muscle cells in the state of sepsis. METHODS: 120 SD rats were randomly divided into 3 groups: 10% group, with 10% of the total cecal length ligated and punctured without use of antibiotic so as to make sepsis model; 30% group, with 30% of the total cecal length ligated and punctured; and control group, undergoing sham operation. Fasting venous blood samples were collected before the operation to detect the fasting plasma glucose (FPG). 0, 8, 16, 24, and 48 hours after the operation 8 rats in each group underwent fasting of food and without fasting of water for 8 hours, i.e., until the 8 th, 16 th, 24 th, 48 th, and 72 nd hours after the operation. Then the rats underwent anesthesia, with blood sample from the vena cava and specimen of gastrocnemius of the hind leg collected, and then killed. The levels of FPG, fasting plasma insulin (FINS), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 were measured. HOMA method was used to calculate the insulin resistance index (Ig-IRI). Immunohistochemistry was used to quantitatively examine the IRS-1 protein and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius. Western blotting and immunoprecipitation were used to semi-quantitatively examine the changes in contents of IRS-1 and its Ser(307) phosphorylation and Tyr phosphorylation in the gastrocnemius respectively. RESULTS: The survival rates at different time points of the control group were all 100%, all significantly higher than those of the other 2 groups (all P < 0.01), and those of the 10% group were all significantly higher than those of the 30% group (all P < 0.01). The levels of TNF-alpha and IL-6 of the 10% and 30% groups at different time points were all significantly higher than those of the control group (all P < 0.01), and those of the 30% group were all significantly higher than those of the 10% group (all P < 0.01). The FPG, FINS, and IgIRI were not significantly different among the 3 groups before the operation, and those of the 10% and 30% groups at different time points after operation were all significantly higher than those of the control group (all P < 0.01) and peaked 8 h after the operation, with those of the 30% group all significantly higher than those of the 10% group (all P < 0.01). The degree of increase of FINS was remarkably higher than that of FPG. IRS-1 was positive and located in the cytoplasm of the gastrocnemius cells in both the control and 30% groups; IRS-1 Tyr phosphorylation was positive in the control group and sporadic positive in the 30% group. IRS-1 Ser(307) was negative in the control group and strong positive in the 30% group. Semi-quantitative examination showed that the IRS-1 level at different time points after operation of the 30% group were not significantly different from those of the control group (all P > 0.05), and IRS-1 Tyr phosphorylation degrees at different time points of the 30% group were all significantly lower than those of the control group (all P < 0.01), and the IRS-1 Ser(307) phosphorylation at different time points of the 30% group were all significantly higher than those of the control group (all P < 0.01). Spearman correlation analysis showed that IgIRI was significantly negatively correlated with IRS-1 Tyr phosphorylation (r = -0.957, P < 0.01), and significantly positively correlated with IRS-1 Ser(307) phosphorylation (r = -0.955, P < 0.01). CONCLUSION: Under the status of sepsis the IRS-1 content in the skeletal muscle cells is unchanged, the level of IRS-1 Tyr phosphorylation level is decreased, and the IRS-1 Ser phosphorylation is increased. The degrees of such changes are closely related with the degree of insulin resistance.